Studies On Isolation And Expression Of Members Of Invertase Gene Family From Citrus

Citrus is one of the most important fruits in China. So far as purchase and sale of fruits in our country is considered, with general increase in yields of all kinds of fruits, the balance has now swung from side of seller's market to that of buyer's market. Following the entry of China into WTO, our domestic citrus will surely face the trade competition from abroad. Therefore, it is very urgent to enhance citrus fruit quality. Sugar content plays an essential part in flavor characteristics of citrus fruit and is also a commercial measure of fresh fruit quality. Moreover, sugar is also the material basis for synthesis of organic acids, cariotenoids, and other nutrient and aromatic ingredients of citrus fruit. Invertase is one of the key enzymes in sucrose metabolism that catalyzes the hydrolysis of sucrose into glucose and fructose. Many enzymes that occurred in higher plants are composed of several isoenzymes. They play important roles in partitioning of assimilates enrout from source to sink organs, control of cell differentiation and plant development, osmoregulation and cell enlargement, adjustment of sugar composition in fruits and storage organs, and plant response to wounding, infection and cold (cold sweetening). Studies of invertase gene family members will provide an important basis for elucidating the mechanism of sugar accumulation and metabolism in fruit and lead the way to scientific regulation and improvement of fruit quality by means of gene engineering.Four pairs of PCR primers designed from some conserved regions of plant vacuolar and cell wall acid invertases were used to screen Citrus sinenesis Lambda genomic DNA library and amplify different fragments of invertase gene from Citrus unshiu, Fortunella crassifolia and Citrus aurantium. Tree-view and homology among seven sequences(nucleic acid and amino acid ) of Citrus invertase gene was analyzed using Seqaid II, Clustalx and Bioedit software package. DIG Southern blots of four members of citrus gene family were analyzed. In addition, DIG northern blots of root, young stem, leaf, flower and young fruit from Fortunella crassifolia and Citrus aurantium were analyzed respectively. The results were summarized below. 1. Citrus sinenesis vacuolar acid invertase gene (CS-VI1) had been isolated by screening Citrus sinenesis Lambda genomic DNA library using Filter Paper Absorption-PCR. The full length ofCS-VIl was 6942bp (including a similar transcription factor, apromoter, ORF and down stream sequence), encoding 588 amino acids. It contained six exons and five introns, and the second exons is the smallest exons in plant kingdom, encoding the so called DPN. Sequence (nucleic acid and amino acid) of C5-F/7 was highly homologous to vacuolar invertase gene of other plants. Therefore, it was supposed to encode a acid invertase located in vacuolar of Citrus sinenesis and had been registered in GenBank, Accession No. is AF433643.2. Citrus sinenesis cell wall invertase gene(CS-CWIJ) had been isolated by screening Citrus sinenesis Lambda genomic DNA library using Filter Paper Absorption-PCR. The sequence of CS-CWI1 had a length of 3073 bp, encoding 281 amino acids and contains two exons and three introns, one of exons being identical to the second exon of CS-VI1, which was the smallest exon in plant kingdom, encoding the so called DPN. Sequence (nucleic acid and amino acid) of CS-CWI1 was highly homologous to cell wall invertase gene of other plants. Therefore, it was supposed to encode an acid invertase located on cell wall of Citrus sinenesis and had been registered in GenBank, Accession No. is AF314197.3. Three fragments of Citrus unshiu invertase gene, one fragment of Fortunella crassifolia invertase gene, one fragment of Citrus aurantium invertase gene had been obtained by PCR. We believe that they belong to putative vacuolar invertase gene l(CU-AIl), ll(CU-AI2) and cell wall invertase gene I (CU-CWI1) of Citrus unshiu, and cell wall invertase gene of Fortunella crassifolia, vacuolar invertase gene of Citrus aurantium respectively. They hav...